Mammalian telomeres are sequential hexameric TTAGGG DNA repeats that cap the ends of linear chromosomes. Structurally, the telomeric DNA forms a lariat, or T-loop, at the end of each chromosome to shield the ends of linear chromosomes from degradation and/or illegitimate recombination. The six-subunit protein complex shelterin binds and protects telomeric DNA with sequence specificity functioning to both inhibit the DNA damage response and regulate T-loop formation. Defects in shelterin function lead to telomere instability and contribute to both premature aging and the progression towards cancer. Recently this protective cap at telomere ends has expanded beyond shelterin to include the telomere repeat- containing RNA, TERRA. TERRA is expressed in several eukaryotes including plants, yeast, fish, and mammals. In human cells, TERRA is transcribed from the C-strand within the subtelomeric region and transcription elongation extends into the telomere repeats generating a transcript between 200bp and 9kb. TERRA is localized within the nucleus and associates with both the telomeric DNA and telomere associated proteins. The discovery of this long non- coding RNA, expressed from a region once considered transcriptionally silent, suggests that there is still much to learn about the mechanisms of telomere maintenance and the role these mechanisms play in the development of cancer. In previous studies, we have demonstrated that TERRA is cell cycle regulated, and that this regulation drives a molecular switch coordinating telomere replication with telomere end protection in vitro. While these studies were among the first to demonstrate a functional requirement for TERRA in telomere maintenance, exactly how TERRA functions to mediate these process in mammalian cells is still unclear. These studies have been limited, in part, by an inability to manipulate endogenous TERRA transcript levels. To overcome this challenge, we have designed a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system that allows us to manipulate gene expression from the endogenous TERRA promoter. Therefore, the goal of this proposal is to further define the function of TERRA in regulation of telomere stability and ultimately, understand how defects in TERRA function contribute to the development of cancer.